Enzymes with the ability to cleave N-acetyl neuraminic acid (NANA), also known as sialic acid, from other carbohydrates are present in many microorganisms. These include bacteria such as Vibrio cholerae, Clostridiun perfringens, Streptococcus pneumoniae and Arthrobacter sialophilus, and viruses such as influenza virus, parainfluenza virus, mumps virus, Newcastle disease virus and Sendai virus. Most of these viruses are of the orthomyxovirus or paramyxovirus groups, and carry a neuraminidase activity on the surface of the virus particles. Many of these neuraminidase-possessing organisms are major pathogens of man and/or animals, and some, such as influenza virus and Newcastle disease virus, cause diseases of enormous importance.
Influenza A and B viruses are major causes of acute respiratory disease, resulting in an estimated 30–50 million infections annually in the United States alone. Influenza A has been responsible for major epidemics, such as the “Spanish Flu” of 1919 which killed millions of people. Influenza remains a difficult disease to control, resulting in significant morbidity, and mortality largely due to secondary infection in eldery or debilitated patients. Vaccines are continually being rendered obsolete by antigenic shift or drift, and consequently immunization is only about 70% effective in preventing infection. Until recently, the only drugs approved by regulatory authorities for treatment of influenza were amantadine and rimantadine, which are ineffective against influenza B, and are known to have serious side-effects. In addition, rapid antiviral resistance to both of these compounds have been shown to be a frequent event with these compounds.
Influenza A and B have two major surface glycoproteins, haemagglutinin (HA) and the enzyme neuraminidase (NA), which are both essential for infectivity. It is believed that HA is necessary for the virus to attach to cells, whereas NA is needed for release of the virus from cell surfaces. There are typically about 600 trimeric HA and about 50 copies of the NA tetramer units on the surface of each virus particle. Both HA and NA therefore are attractive potential targets in the search for anti-influenza drugs.
Influenza virus haemagglutinin binds to the sialic acid-containing glycoproteins and glycolipids on cell-surface receptors, thereby initiating the process of attachment of the virus to a cell and subsequent infection. The strength of the binding of a virus particle to the cell membrane appears to depend on the interaction of multiple copies of the influenza HA with multiple sialic acid groups on the cell surface.
Using this concept of a polyvalent interaction (Mammen et al., Angew. Chem., 1998 37: 2754–2794), several workers have reported the synthesis of macromolecules containing two or more sialic acid derivatives which act as haemagglutinin inhibitors. Although some strong HA inhibitors have been discovered, none of these polyvalent macromolecules has been shown to prevent influenza infection in vivo. Recent papers by Whitesides and other workers (J. Amer. Chem. Soc., 1996 118 3789–3800; J. Medicinal Chem., 1995 38 4179–4190; Bioconjugate Chemistry, 1999 10: 271–278) have summarised the various attempts to use this approach to the design of inhibitors of influenza haemagglutinin.
It has long been thought that inhibitors of neuraminidase might prevent infection by neuraminidase-bearing viruses. Most of the known neuraminidase inhibitors are analogues of neuraminic acid, such as 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) and some of its derivatives (Meindl et al, Virology, 1974 58 457). Our International Patent Publication No. WO 91/16320 describes a number of analogues of DANA which are active against viral neuraminidase, and it has been shown in particular that 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Compound (A), code number GG167) is useful in the treatment of influenza A and B (N. Engl. J. Med., 1997 337 874–880). Other patent applications describe various closely-related sialic acid derivatives (eg. PCT Publications No. WO95/18800, No. WO 95/20583 and No. WO 98/06712), and anti-viral macromolecular conjugates of GG167 have also been described (International Patent Application No. PCT/AU97/00771, WO98/21243).

In addition to the sialic acid-based inhibitors mentioned above, other types of highly active inhibitors of influenza virus neuraminidase have also been described, particularly those based on 5- and 6-membered carbocyclic ring systems (eg. International Patent Publications No. WO96/26933 and No. WO97/47194). Despite intense research activity, to date only two anti-influenza drugs which act at the NA site, “Relenza” (Trade mark of Glaxo Wellcome plc) and “Tamiflu” (Trade mark of Hoffman-LaRoche AG) have been approved for clinical use. Other neuraminidase inhibitors have been disclosed in WO99/33781, specifically (1S,2S,3R,4R)-3-[(IR)-1-(acetylamino)-2-ethylbutyl]-4-{[amino(imino)methyl]amino]-2hydroxycyclopentane carboxylic acid, which is in phase III clinical trials.
Recently, International Patent Publication No. WO97/06157, No. WO98/06712 and European Patent Application No. 0823428 have described certain derivatives of compound (A) in which the normal sialic acid 7-hydroxy group is replaced by various other functionalities, which inhibit multiplication of the influenza virus.
International Patent Publication Nos. WO95/34595 and WO98/03572, by Biomolecular Research Institute Ltd, respectively disclose dendrimers having a plurality of terminal groups and linear polymers having a plurality of side chain groups. In WO95/34595, the terminal groups may inter alia be anionic, and may contain a carboxylic acid group. Sialic acid and related compounds are neither disclosed nor suggested. In WO98/03572, the side chain groups may be sialic acid or a derivative thereof; the only example of such a compound utilises linkage of the sialic acid moiety via a sulphur atom at the 2-position, in line with the conventional wisdom at that time. The use of 2,3-dehydrosialic acid derivatives as side chain groups is neither disclosed nor suggested.
In our International patent application No. PCT/AU00/00165 filed on Mar. 9, 2000, we showed that when two neuraminidase-binding compounds are suitably linked together through a region of the molecule that is not involved in binding to the active site, the resultant dimers show enhanced anti-viral activity. In particular we have found that, although an extra substituent attached to compound (A) at the 7-position generally causes a slight decrease in the anti-influenza potency, when two such 7-substituted molecules of compound (A) are both attached to a suitable spacer moiety, the anti-influenza activity can be significantly improved. Though not wishing to be bound or limited by any proposed mechanism for the observed effect, we believe that the dimeric compounds have improved anti-influenza activity because they may be able to bind to two separate neuraminidase molecules, and thereby cause aggregation of the neuraminidase tetramers and/or the influenza virions, or that by having one copy of zanamivir bound to the active site of the neuraminidase, and a second copy in close proximity, then the binding kinetics may be more efficient, in that as one copy dissociates the second copy can bind more rapidly than a free molecule of zanamivir. We have now shown that multivalent neuraminidase inhibitor conjugates, ranging up to dendrimer-type molecules, have longer duration of action. Again without wishing to be bound by theory, the basis for the long residency time in the lungs is thought to be due to the size and molecular weight of the macromolecule preventing entry through tight junctions in the respiratory epithelium, and the polarity of the macromolecule being such that passage through the cell membranes occurs very inefficiently. An alternative theory is that the compounds themselves interact with the phospholipids in the cell membrane or other components of the respiratory epithelium, and increase the residency time in the lungs.
It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.